Therapeutic composition and use

ABSTRACT

A pharmaceutical dispenser, comprising a liquid formulation comprising nitric oxide, and means for forming a nebulised mist of the liquid formulation. The nebulised mist of the liquid formulation is used primarily in the treatment of respiratory diseases.

The present invention relates to the use in therapy of nitric oxide.

There has been much interest in recent years in pharmaceuticalapplications of nitric oxide [NO] and nitric oxide precursors. Nitricoxide is a potent vasodilator which is synthesised and released byvascular endothelial cells and plays an important role in regulatingvascular local resistance and blood flow. In mammalian cells, NO isprincipally produced along with L-citrulline by the enzymatic oxidationof L-arginine. Nitric oxide is also involved in the inhibition of bothplatelet and leucocyte aggregation and adhesion, the inhibition of cellproliferation, the scavenging of superoxide radicals and the modulationof endothelial layer permeability. Nitric oxide also has been shown topossess anti-microbial properties, reviewed by F. C. Fang (1997) (J.Clin. Invest. 99 (12) 2818-2825 (1997)).

In addition to internal cell-mediated production, NO is also continuallyreleased externally from the surface of the skin by a mechanism whichappears to be independent of NO synthase enzyme. Nitrate excreted insweat is reduced to nitrite by an unknown mechanism, which may involvenitrite reductase enzymes, which are expressed by skin commensalbacteria. Alternatively mammalian nitrite reductase enzymes may bepresent in the skin which could reduce nitrite rapidly to NO on the skinsurface.

The production of NO from nitrite is believed to be through thefollowing mechanism:NO₂ ⁻+H⁺

HNO₂   [1]2HNO₂

N₂O₃+H₂O  [2]N₂O₃

NO+NO₂   [3]

There are a number of disclosures dealing with the artificial provisionof NO.

WO 95/22335 (Benjamin & Dougal) discloses a dosage form for thetreatment of bacterial, virus, or fungal conditions which comprises apharmaceutically acceptable acidifying agent, a pharmaceuticallyacceptable source of nitrite ions or a nitrate precursor therefor, and apharmaceutically acceptable carrier or diluent, wherein the acidifyingagent is adapted to reduce the pH at the environment of use to below pH4. Preferably the acidifying agent is an organic acid, for examplesalicylic acid or ascorbic acid. The precursor for the nitrite ion maybe an alkaline metal or alkaline earth metal capable of conversion to anitrate by enzymatic action. In a particularly preferred form of theinvention the acidifying agent and the source of nitrite ions orprecursor therefore are separately disposed in said cream or ointmentfor the admixture to release nitrite ions at the environment of use.Alternatively an acid composition may be presented for administration intablet or liquid form.

U.S. Pat. No. 5,648,101 (Tawashi) discloses a method for delivering NOgas to a desired site or into the body of a sentient animal, e.g.humans, comprising combining and causing to react a soluble reducingsalt, preferably ferrous sulphate, and a nitrite, preferable sodiumnitrite in the presence of moisture in situ at or adjacent to such asite. Means for such delivery include compositions such as tablets,capsules, ointments, creams, lotions and sprays containing mixtures ofparticles or granules of the two reactants, transdermal patches andosmotic pumps for combining solutions of reactant or reactants in situ.Wink et al, The role of nitric oxide chemistry in cancer treatment,(Biochemistry (Moscow) 802-809; 63(7):1998) discloses the effect ofnitric oxide upon mammalian tumours. Current disclosures in the field ofcancer treatment refer to endogenous production of nitric oxide.Attempts to increase local availability have been limited to non-directinterventions such as dosing with nitric oxide precursors (L-arginine)and manoeuvres to increase the half-life/bioavailability of endogenousnitric oxide by temporarily modulating breakdown pathways.

Other clinical methods involving the use of NO precursors are disclosedin WO-A-99/02148, WO-A-95/09612 and Chemical Abstracts; 127:130755, B.H. Cuthbetson et al, British Journal of Anaesthesia, (1977), 78(6),714-717.

The topical use of gaseous nitric oxide and nitric oxide precursors asantimicrobials is also known. WO-A-01/53193 discloses the use ofacidified nitrite to produce nitric oxide topically at the skin surface.The treatment is useful in the treatment of ischaemia and relatedconditions.

In topical application to the sldn of nitrite at concentrations of up to4% in an inert carrier cream or ointment, the nitrite, when mixed withan organic acid such as ascorbic acid (vitamin C), reacts to produceoxides of nitrogen to cause the release of nitric oxides leading tosustained vasodilation of the microcirculatory blood vessels, withoutsignificant inflammation.

Useful reviews of the use of NO in therapy are provided in the followingreview articles; Chemical Abstracts; 134:216558, W. E. Hurford et al,Nitric Oxide, (2000), 931-945; Chemical Abstracts; 128:21192, M.Andresen et al, Revista Medica de Chile, (1997), 125 (8) 934-938;Chemical Abstracts; 124:44545, M. Beghetti et al, Expert Opinion onInvestigational Drugs, (1995), 4 (10) 985-995.

All these previous proposals for the clinical application of NO havefocussed on the use of either gaseous NO, or else of NO precursors insolution, suspension, or topical preparations.

We have now found that, surprisingly, when nitric oxide is dissolved orsuspended in a liquid, either by passing gaseous NO through a liquid, orby generating NO in situ in the liquid, clinically significantconcentrations can be established either dissolved or dispersed withinthe liquid, even though a significant proportion of the NO generated inor passed through the liquid gas is released immediately to theatmosphere. For example, when NO is produced in aqueous solution by thereaction of 0.5 molar nitrite with 0.5 molar citric acid, theconcentration of NO dispersed or dissolved in the liquid formulation,after gas evolution, has been found to be of the order of 1,500 ppb (1.5ppm) Additionally, the resulting liquid formulation (referred to hereinfor simplicity as simply a “solution” even though in practice the NO maybe present both in true solution, and in the form of a dispersion orsuspension) remains stable within a time span required to performtherapeutic manoeuvres and elicit biological actions (for example, forperiods in excess of one hour). Accordingly, liquid formulations areeffective for the treatment of a number of clinical conditions, byadministration of a nebulised spray of liquid formulation.

Accordingly, in a first aspect of the invention there is provided apharmaceutical dispenser, comprising

-   -   a liquid formulation comprising a clinically effective        concentration nitric oxide, or    -   means for forming such a liquid formulation from a nitric oxide        source, and    -   means for forming a nebulised mist of the liquid formulation

The nitric oxide may be present in the liquid formulations according tothe invention in true solution, and/or in the form of a dispersion orsuspension (for example in colloidal suspension). All such formulationtypes are referred to herein as “solutions”.

By “nebulised mist” as used herein is meant any form in which the liquidformulation may be sprayed as a mist of droplets for application, suchas those conventionally produced by nasal sprays or hand-heldapplicators used for treating respiratory conditions such as asthma.

In such liquid formulations, the effective compositions will generallycontain concentrations of dispersed and/or dissolved NO in the range offrom 10 to 40,000 ppb (parts per billion) by weight, preferably from 100to 10,000 ppb, more preferably from 1,000 to 10,000 ppb.

The liquid formulations employed in accordance with the presentinvention may be prepared by the use of a pharmacologically acceptableacidifying agent, together with a pharmacologically acceptable source ofnitrite ions or a nitrite precursor.

In accordance with the present invention active liquid formulations ofnitric oxide as described above may administered by means of a nebuliserand employed as therapeutic compositions for the treatment of arespiratory disease, for example, bronchiectasis, allergic bronchiapulmonary aspergillosis, Chlamydia pneumonia, obstructive pulmonarydisease, Bacillus anthracis, Streptococcus pneumoniae, mycobacterium,Mycobacterium tuberculosis, M. bovis, M africanum, acute respiratorydistress syndrome, occupational lung disease, allergic lung disease, orimpaired respiratory function, RSV Bronchiolitis, precipitateexacerbations of asthma, chronic obstructive pulmonary disease (COPD),viral pneumonia, pneumonia, tuberculosis, acute respiratory distresssyndrome (ARDS), acute lung injury, hypoxemic respiratory failure, andasthma.

In a particular aspect of the invention, the nitric oxide solution maybe employed for the treatment of anthrax.

In particular, the composition may be contained within a delivery systemwhich allows the production of a nebulised mist of the nitric oxideliquid formulation which can be passed to the lungs of a human or animalpatient.

A further aspect of the invention provides the use of such liquidformulations of nitric oxide in the preparation of a therapeuticcomposition for the treatment of cancer, wherein the composition isadapted for administration by means of a nebuliser.

The liquid mixture may be made by the combination of nitrite with anorganic acid such as ascorbic acid in a liquid medium, such as water or,more preferably, physiological saline. Alternatively, it can be made bystreaming pure nitric oxide gas through the liquid medium in order toform the liquid formulation.

The pH of the resulting liquid mixture may be manipulated by thetitration of the acidifying agent and/or subsequent chemical bufferingusing standard techniques to create a pharmaceutically acceptableformulation for application to the respiratory system. The pH in thelungs is approximately 7.4 and therefore the pH of the resulting liquidmixture is preferably approximately 7.4.

The nebuliser and nebulisation method to be used in accordance with thepresent invention may be any conventional method used in general medicalor veterinary practice, which is able to produce a nebulised spray witha particle size in the respiratory range, as disclosed for example in J.Heyder et al., (“Deposition of particles in the human respiratory tractin the size range 0.0005 to 15 microns” Jnl. of Aerosol Science 1989 pp1-21). The preferred particle size of the nebulised particles is in therange of from 0.1 to 10 micrometer, preferably from 2 to 5 micrometer.The particle size can be adjusted in order to target the dose to thedesired region of the respiratory tract, as is disclosed in the Heyderet al paper referred to above.

The nebuliser employed may, for example, be of the pressurised dispensertype widely used for the oral or nasal administration of anti-asthmadrugs and the like.

The acidifying agent may include any suitable organic acid such asascorbic acid (vitamin C), salicylic acid, acetyl salicylic acid, aceticacid or a salt or a derivative thereof, generally in a concentration upto 20% w/v, preferably 0.25 to 10% w/v, more preferably 4 to 6% w/v. Aparticularly preferred concentration is 4% or 5% w/v. Other acidifyingagents include but are not limited to, ammonium or aluminium salts,phenol, and benzoic acid. Inorganic acids such as hydrochloric acid maybe used if sufficiently dilute and/or appropriately buffered. Theacidifying agent may be present as a dissolved salt or in a liquid form.

The pharmacologically acceptable source of nitrite ions may an alkalinemetal nitrite or an alkaline earth metal nitrite, for example, LiNO₂,NaNO₂, KNO₂, RbNO₂, CsNO₂, FrNO₂, Be(NO₂)₂, Mg(NO₂)₂, Ca(NO₂)₂,Sr(NO₂)₂, Ba(NO₂)₂, or Ra(NO₂)₂. Alternatively, a nitrite precursor maybe used as the source of the nitrite ions in the composition, such asfor example a dilute solution of nitric acid. Other sources of nitriteions are nitrate ions derived from alkali metal or alkaline earth metalsalts capable of enzymic conversion to nitrite, For example, LiNO₃,NaNO₃, KNO₃, RbNO₃, CsNO₃, FrNO₃, Be(NO₃)₂, Mg(NO₃)₂, Ca(NO₃)₂,Sr(NO₃)₂, Ba(NO₃)₂, or Ra(NO₃)₂. The concentration of the nitrate ionsource prior to acidification may be up to 20% w/v, suitably 0.25 to10%, preferably 4 to 6%. A particularly preferred concentration is 4% or5% w/v.

The composition employed in accordance with the invention is preferablysaturated with nitric oxide in solution.

The pharmaceutical composition may then administered by nebuliser, forexample as described by Heyder above.

Dosages of nitric oxide for the purposes of the invention can varywithin wide limits, depending upon the disease or disorder to betreated, the severity of the condition, and the age and health of theindividual to be treated. A physician will readily be able to determineappropriate dosages to be used.

This dosage may be repeated as often as appropriate. If side effectsdevelop the amount and/or frequency of the dosage can be reduced orotherwise altered or modified, in accordance with normal clinicalpractice.

Compositions may be formulated according to the invention for human orfor veterinary medicine. The present application should be interpretedas applying equally to humans as well as to animals, unless the contextclearly implies otherwise.

The composition has important clinical benefits, either alone or inconjunction with other anti-infective therapy in, for example, thefollowing conditions:

1. Bronchiectasis: a relatively common lung condition in which chronicsepsis occurs in areas of the lung. A particular example of this is theinherited lung disease, cystic fibrosis.

2. Allergic bronchia pulmonary aspergillosis: a complication of asthmain which patients become allergic to the fungus aspergillus whichcolonises their airways and causes lung damage and bronchia actisis.

3. Chlamydia pneumonia; It has been suggested that some cases of lateonset asthma may be related to chronic infection of the airways withChlamydia pneumonia.

4. Obstructive pulmonary disease; Approximately 30% of chronicobstructive pulmonary disease (COPD patients) have bacterialcolonisation with streptococcus pneumoniaea and/or haemophulusinfluenzae. This is difficult to eradicate and there is evidence thatbacterial colonisation leads to exacerbations of COPD and worsensprognosis.

5. Mycobacterium; Chronic infection with atypical mycobacterium such asmycobacterium xenopi, mycobacterium chelonae and mycobacterium fortuitummay occur in previously damaged lungs and it is difficult to eradicate.

In addition, the nebulised mist of nitric oxide liquid formulation, wheningested into the lungs or nasal tract, is advantageous in the treatmentof, for example Bacillus anthracis, Mycobacterium tuberculosis, M.bovis, M. africanum; acute respiratory distress syndrome; occupationallung diseases; allergic lung diseases; impaired respiratory function andassociated conditions.

Although not wishing to be bound by any theory of operation, it isbelieved that the presentation of nitric oxide in liquid formulationform results in preferential take-up of the nitric oxide by theinfectious organisms, rather than take-up by blood haemoglobin leadingto inactivation.

There is a range of acute lung infections, which may be shortened withthe application of the invention; these include viral infections, whichcan either cause acute illness themselves e.g.

1. RSV Bronchiolitis in children or precipitate exacerbations of asthmaor chronic obstructive pulmonary disease (COPD)

2. Viral pneumonia such as influenza pneumonia, for which at presentthere is nothing except supportive treatment

3. Pneumonia

4. Tuberculosis

The liquid formulation of the present invention may be used acutely inconjunction with other therapies to help eradicate these diseases or toprevent re-infection or decrease the infective load. By alteringparticle size, it is possible to target the aerosol to the largeairways, smaller airways or alveoli in order to aid the efficacy in thetreatment of disease.

The nebulised system is advantageous in comparison to the use ofrespiratory nitric oxide gas. For example, hypoxic pulmonaryvasoconstriction, a protective phenomenon causing vasoconstriction ofpoorly ventilated lung units, is a normal response toventilation/perfusion mismatch. The use of inhaled nitric oxide gas hasbeen suggested to modify this normal response, resulting invasodilatation of poorly ventilated areas, increased shunting throughthe lung and worse oxygenation. Delivery of nitric oxide in fluidmixture by nebulisation will reduce this risk as the active elements ofthe system are only delivered to functioning lung units. Also, becauseof its short half-life, inhaled NO gas must be delivered continuouslyand abrupt withdrawal of therapy can be associated with life threateningrebound hypoxemia. This system provides an aqueous, slower release formof NO to the various regions of the respiratory tract. Furthermore, thesystem offers novel and significant advantages because of simplifieddelivery technology and intermittent instead of continuous therapy.

The nebulised system also does not have the problems inherent in systemsinvolving the dosage, instillation or nebulisation of NO/nucleophileadducts, S-nitrosothiols, diethylamine-NO complexes (DEA/NO)S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione (GSNO),SIN-1 and similar systems whereby the associated carriers such asnucleophiles and break down products may be associated with potentiallocal or systemic toxicity.

In addition to the anti-microbial functions of the invention, otherknown biological functions of the nitric oxide molecule may also bevariably induced by the nebulised system, for example vasodilatation.The vasodilator effects may be utilised in the treatment of respiratorydisease, such as but not limited to acute respiratory distress syndrome(ARDS), acute lung injury and hypoxemic respiratory failure. Whenapplied to the treatment of asthma (chronic inflammation leads to theclinical use of corticosteroids which reduce/eliminate intrinsic NOproduction potentially leading to vasoconstriction and increased risk ofinfection) the nebulised system may be used as a vasodilatoryreplacement therapy, either alone or in combination with othertreatments (for example as prostanoid therapy).

The vasodilator effects to the system may also be utilised in thetreatment of cardiovascular disease (for example, but not limited topulmonary hypertension, angina and coronary heart disease) in theabsence of significant systemic vasodilation.

The nebulised composition may also be used in the management of sicklecell disease. The two most common complications are vaso-occlusiveepisodes and acute chest syndrome [ACS]. ACS is a leading cause ofmortality and affects 50% of patients at least once in their lifetime.Although risk factors for ACS have not been well defined, pulmonaryfunction tests in patients with sickle cell disease identifyhyper-reactivity of the airways as a modifiable risk factor. Thevasodilator and anti-microbial properties of the nebulised compositionare particularly relevant to the therapy of the patients. In addition,concomitant treatment by the addition of corticosteroids such asdexamethasone to the nebulised composition may be efficacious in somepatients.

Nitric oxide also modulates tissue oxygenation via changes in membranepermeability.

Nitric oxide exerts a potent inhibitory effect on both platelet andleucocyte aggregation and adhesion.

Augmentation of ciliary function is especially relevant in themanagement of cystic fibrosis and other pathological conditionsassociated with dyskinesia of ciliary function. Airway cilia have shownciliary-beat-frequency stimulation by cyclic guanosine monophosphate andendogenous prostaglandin E₂. The nebulised delivery of nitric oxide tothe respiratory system by the disclosed system increases concentrationsof guanosine monophosphate and leads to the release of prostaglandin E₂and direct action.

Furthermore, the system may additionally be combined with Primarypharmaceuticals such as corticosteroids, surfactants, hormones (such asinsulin), antibiotics (such as colomycin and tobramycin), prostanoids(such as prostacyclin) and cytotoxic agents to produce an increasedefficacy of delivery, absorption and action.

Nitric oxide delivered in a liquid formulation as disclosed herein maybe used therapeutically as an adjunct to chemotherapy by increasingefficacy of delivery, absorption and action of cytotoxic agents (forexample the cisplatin, melphalan, tamoxifen, paxitaxol, and anastrozole)to the respiratory system and directly modulating apoptosis. Increasedsensitivity of tumour cells is mediated by nitric oxide due to theinhibition of key DNA repair proteins such as DNA ligases. One of themajor factors that limits the effectiveness of radiation therapy is thepresence of radioresistant hypoxic tumour cell populations. Nitric oxidecan radiosensitise mammalian hypoxic cell populations and is at least aseffective as oxygen in this role. Carbon-centred radicals are initiallygenerated by ionising radiation on DNA. In the absence of nitric oxideor oxygen, these reactive radicals scavenge nearby protein hydrogenatoms, thereby limiting the number of DNA lesions per photon. The nitricoxide reacts with these high-energy complexes and inhibits the repairmechanism from abstracting protein hydrogen atoms, which would normallyfacilitate DNA repair. The effects of this inhibition result in theradiosensitising of the hypoxic cells. The fixation of radiation induceddamage increases the number of lesions per photon. Nitric oxide has ahigher diffusion coefficient than oxygen and therefore penetratesfurther into the tissues. The physiological functions of nitric oxidemolecules additionally modulate the response to therapy.

Both the radiotherapy and the cytotoxic drug delivery application may beachieved by nebulisation of the system, infiltration or by directinjection of the tumour or surrounding locus.

In addition to administration to the respiratory tract, the nitric oxidefluid mixture may be administered to any part of the human or animalbody to treat or prevent microbial infection. For example, the mixturemay be sprayed onto the skin to treat dermatological conditions.Alternatively, the mixture may be administered by spraying internalorgans of the body, for example during and after operations, to preventmicrobial infection.

The nitric oxide liquid formulation described may be additionally usedin non-medical areas such as cosmetics and beauty therapy, in view ofits biological effects upon skin blood flow and tissue oxygenation.Areas of application may include but are not limited to the treatment ofcellulite, stretch marks and skin blemishes. When combined withauxiliary agents, such as minoxidil for hair growth, the penetration andefficacy of the agents is enhanced.

The nitric oxide fluid mixture system may exert positive and fundamentalbiological influences upon the normal physiological functions ofspermatozoa (including motility, progression, capacitation, acrosomereaction and zona binding), fertilisation, embryo development,implantation and early development. The system may also haveimplications in the understanding and management of a wide range ofgynaecological systems such as menstruation and pathological conditions.The influences of NO on gynaecology and fertility are exerted in abiphasic dose dependant function.

The nitric oxide fluid mixture may additionally be applied to inanimateobjects for the purposes of sterilisation, and in order to preventcolonisation by microorganisms.

A viscosity modifier, for example propylene glycol or other knownpharmaceutically acceptable substance with viscosity modifyingproperties may be employed in order to increase the adherence of thecomposition to a surface to be treated.

The invention will now be described, by way of illustration only withreference to the following examples and figures which are provided forthe purposes of illustration and are not to be construed as beinglimiting on the invention.

FIG. 1 shows the anti-microbial properties of the NO-generation gel atdifferent nitrite ion concentrations against Staphylococcus aureusNCTC9353 and Escherichia coli NCTC10148 at twenty-four hours exposure.The vertical axis shows microbial survival as a percentage and thehorizontal axis shows NO-gel concentration in mM.

FIG. 2 shows the anti-microbial properties of the NO-generation gel atdifferent nitrite ion concentrations against MRSA (NCTC11561) at fourhours exposure. The vertical axis shows microbial survival as apercentage and the horizontal axis shows Nitrite concentration inpercentage.

In both FIGS. 1 and 2, the values shown are medians (n=3).

EXAMPLE 1

A liquid composition containing nitric oxide was prepared as follows. 10ml of 0.5 M sodium nitrate aqueous solution was added to 10 ml of 0.5 Mascorbic acid aqueous solution. The liquid composition was mixed inorder to liberate nitric oxide. After liberation of nitric oxide, theliquid composition was diluted in the ratio of 1:5 with distilled water.The resulting liquid composition was then tested using a World PrecisionInstruments NO Nanosensor ISO-NOPNM. The system uses a 100 nm diametertip with optimal detection limit of less than 0.5 nm. The assay andcalibration were carried out in accordance with the World PrecisionInstruments instruction manual. The liquid composition was found to havean nitric oxide concentration of approximately 300 ppb by weight, and tobe stable for a period in excess of one hour.

By varying the conditions of preparation, it was found that otherconcentrations of NO in the final solution may be achieved.

Examples 2 and 3 are not examples of the invention but are included todemonstrate the effectiveness of NO in treating microorganisms.

EXAMPLE 2 Transmembrane Anti-microbial Properties of NO-generation Gel

The antimicrobial properties of NO-generation gel after diffusionthrough a 10 μm Sympatex™ membrane were investigated as follows. NO wasgenerated by an admixture of sodium nitrite and ascorbic acid in 0.8%agar gel, using 1% sodium chloride as an intermediate. The preparationwas tested on S. aureus NCTC9353 and E. coli NCTC10148 using a range ofconcentrations of sodium nitrite and ascorbic acid. Cultures of S.aureus and E. coli were prepared by inoculating 20 ml of LB(Luria-Bertani 10 g Bacto-Tryptone, 5 g Bacto-Yeast extract and 10 g/lsodium chloride at pH 7.5) broth with 2-3 colonies, and incubated at 37°C. overnight. 24 ml of 1.5% agar in NaCl were inoculated with 1 ml ofeither S. aureus or E. coli and poured into Petri dishes. Discs ofmembrane (100 mm in diameter) were sterilised in 70% ethanol and thediscs were then placed in a laminar flow cabinet to allow the ethanol toevaporate. 5 ml of 0.8% agar in 1% NaCl, containing either sodiumnitrite or ascorbic acid at final concentrations of 500 mM, 250 mM, 165mM, 50 mM, 25 mM, 5 mM, 2.5 mM and 0.5 mM were prepared. Finalconcentrations in use are halved.

In the centre of sterile inverted Petri dish lids, 1 ml of eachconcentration of sodium nitrite and ascorbic acid was added and mixed.Disinfected membrane was then placed over the top of this immediately,using sterilised forceps. The membrane was carefully positioned so thatit hung over the edge of the lid equally in all directions. The base ofthe Petri dish was then placed upside down on top of thelid/mixture/membrane arrangement ensuring that a 2-3 mm gap was leftbetween the membrane and the inverted inoculated agar.

The apparatus was incubated overnight at 37° C. after which it wasremoved. The base of the Petri dish (upside down) was removed and thecentral area of agar sampled by cutting a circle using a sterile plasticmeasuring cup. The agar was then macerated in 10 ml of LB broth and 5 mlof sterile glass beads. Serial dilutions were carried out and thesamples plated onto blood agar plates that were incubated for 24 hoursat 37° C. The surviving colonies were then counted.

Anti-microbial properties of nitric oxide were seen at concentrations ofnitrite above 50 mM. Below this concentration partial or noanti-microbial activity was seen. Above this concentration, cell lysiswas complete resulting in complete killing of the bacteria. The resultsshown in FIG. 1 illustrate the anti-microbial effect of varyingconcentrations of NO-generation gel and resulting diffusion throughSympatex™ 10 μm membrane.

Similar experiments have demonstrated the effectiveness of compositionscontaining low concentrations of NO against Bacillus anthracis.

EXAMPLE 3 Transmembrane Anti-microbial Properties of NO-generation Gel

The antimicrobial properties of NO-generation gel after diffusionthrough a 10 μm Sympatex™ membrane were investigated as follows. NO wasgenerated by an admixture of sodium nitrite and ascorbic acid in 0.8%agar gel, using 1% sodium chloride as an intermediate. The preparationwas tested on MRSA (NCTC11561) using a range of concentrations of sodiumnitrite and ascorbic acid. Cultures of MRSA (NCTC11561) were prepared byinoculating 20 ml of LB (Luria-Bertani 10 g Bacto-Tryptone, 5 gBacto-Yeast extract and 10 g/l sodium chloride at pH 7.5) broth with 2-3colonies, and incubated at 37° C. overnight 25 ml of 1.25% agar in 0.8%NaCl were inoculated with 1 ml bacterial suspension and poured intoPetri dishes. Discs of membrane (100 mm in diameter) were sterilised in70% ethanol and the discs were then placed in a laminar flow cabinet toallow the ethanol to evaporate. 5 ml of 0.8% agar in 1% NaCl, containingsodium nitrite or ascorbic acid were prepared at a range ofconcentrations.

In the centre of sterile inverted Petri dish lids, 1 ml of eachconcentration of sodium nitrite and ascorbic acid was added and mixedproducing a final concentration range (1500 mM, 725 mM, 362 mM, 181M, 91mM, 45 mM, and 23 mM). Disinfected membrane was then placed over the topof this immediately, using sterilised forceps. The membrane wascarefully positioned so that it hung over the edge of the lid equally inall directions, The base of the Petri dish was then placed upside downon top of the lid/mixture/membrane arrangement ensuring that a 2-3 mmgap was left between the membrane and the inverted inoculated agar.

The apparatus was incubated for four hours at 37° C. after which it wasremoved. The base of the Petri dish (upside down) was removed and thecentral area of agar sampled by cutting a circle using a sterile plasticmeasuring cup. The agar was then macerated in 5 ml of phosphate bufferedsolution and sterile glass beads. Serial dilutions were carried out andthe samples plated onto CLED (Cysteine lactose electrolyte deficiency)agar plates that were incubated for 24 hours at 37° C. The survivingcolonies were then counted.

Anti-microbial properties of nitric oxide were seen at concentrations ofnitrite above 1.5% nitrite concentration. Below this concentrationpartial or no anti-microbial activity was seen. Above thisconcentration, cell lysis was complete resulting in complete killing ofthe bacteria. The results shown in FIG. 2 illustrate the anti-microbialeffect of varying concentrations of NO-generation gel and resultingdiffusion through Sympatex™ 10 μm membrane.

The foregoing Examples are provided by way of guidance only, and variousother possibilities will be apparent to one of skill in the art, withinthe scope of the appended claims.

1. A pharmaceutical dispenser, comprising a liquid formulationcomprising a clinically effective concentration of nitric oxide, ormeans for forming such a liquid formulation from a nitric oxide source,and means for forming a nebulised mist of the liquid formulation. 2-13.(canceled).
 14. A method of treatment of a respiratory diseasecomprising: producing a liquid formulation comprising a clinicallyeffective concentration of nitric oxide dissolved and/or dispersedtherein; forming a nebulised mist of said liquid formulation; andspraying said nebulised mist onto a site in the respiratory tractrequiring treatment.
 15. A method as claimed in claim 14, wherein therespiratory disease is a microbial disease.
 16. A method as claimed inclaim 14, wherein the respiratory disease is bronchiectasis, allergicbronchia pulmonary aspergillosis, Chlamydia pneumonia, obstructivepulmonary disease, Bacillus anthracis, Streptococcus pneumoniae,Mycobacterium tuberculosis, M. bovis, M. aricanun, acute respiratorydistress syndrome, occupational lung disease, allergic lung disease, orimpaired respiratory function, RSV Bronchiolitis, precipitateexacerbations of asthma, chronic obstructive pulmonary disease (COPD),viral pneumonia, pneumonia, tuberculosis, acute respiratory distresssyndrome (ARDS), acute lung injury, hypoxemic respiratory failure, orasthma.
 17. A method as claimed in claim 14 wherein the liquidformulation additionally comprises a buffer.
 18. A method as claimed inclaim 14, comprising the additional step of providing a source of nitricacid for generating the nitric oxide in the liquid formulation.
 19. Amethod as claimed in claim 18, wherein the source of nitric oxidecomprises a pharmacologically acceptable source of nitrite ions or anitrite precursor and a pharmacologically acceptable acidifying agent.20. A method as claimed in claim 14, wherein the liquid formulation isan aqueous solution.
 21. A method as claimed in claim 14, wherein theliquid formulation contains from 10 to 40,000 parts per billion byweight of NO.
 22. A method as claimed in claim 21, wherein the liquidformulation contains from 100 to 10,000 parts per billion by weight ofNO.
 23. A method as claimed in claim 14, wherein the liquid formulationalso comprises an additional pharmaceutically active component.
 24. Amethod as claimed in claim 23, wherein the additional pharmaceuticallyactive component comprises a corticosteroid, a surfactant, a hormone, anantibiotic, a prostanoids, or a cytotoxic agent.